System and method for cell culture scaling

ABSTRACT

The present set of embodiments relate to a bioproduction system, method, and apparatus for creating a scalable bioreactor system. Specifically, the present set of embodiments enable the determination of bioreaction performance characteristics of a commercial scale by matching operational parameters between a small test scale bioreaction to that of a commercial scale bioreaction. The system and methods do not rely on simply making bioreactor apparatuses across scales the same dimensionally which would not account for differences in fluid dynamic properties between very small to very large volumes, but requires tuning of a variety of systems (mixing assembly, sparger system, and headspace airflow system) in conjunction with one another to achieve predictive outcomes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/712,343, filed Jul. 31, 2018, U.S. Provisional Application No.62/670,934, filed May 14, 2018, and U.S. Provisional Application No.62/618,215, filed on Jan. 17, 2018, which are both incorporated hereinby specific reference.

BACKGROUND

The biopharmaceutical industry uses a broad range of mixing systems fora variety of processes such as in the preparation of media and buffersand in the growing, mixing and suspension of cells and microorganisms.Some conventional mixing systems, including bioreactors and fermenters,comprise a flexible bag disposed within a rigid support housing. Animpeller is disposed within the flexible bag and is coupled with thedrive shaft. Rotation of the drive shaft and impeller facilitates mixingand/or suspension of the fluid contained within flexible bag.

Scientist and engineers have worked to create stirred tank reactors thatcan provide not only an aseptic and well controlled environment for cellculture growth, but also deliver a robust scale-up solution fromresearch to manufacturing scale. Traditionally good engineeringprinciples have been applied in such a manner to design reactors basedupon linear geometric scaling approach (i.e. the vessels will havesimilar height/diameter ratios as well as similar impeller/tank diameterratios). This works fairly well in many cases, but the operators usuallyhave to make many complex decision as how best to choose operationalsettings in order to have reproducible results through scale-up. Becauseit is a two phase and metabolically based system, most of theoperational parameters interact dynamically and are often non-linear inresponse; the best parameter choices and process outcome are often fartoo unpredictable, costly, and time consuming to be addressed properlyat the operator level.

Those skilled in the art of biopharma manufacturing recognize theimportance of having a good scale-up and scale-down models that are ableto simulate or ideally replicate the physiological growth conditionsfound in large-scale bioreactor. The reason for the scale-down model'simportance is often related to the need to screen multiple parametersquickly (optimization of cell clone, media, critical operatingparameters, or product quality) and it is beneficial to be able toperform a wide range of experiments under well controlled conditions atlower cost and with less labor. A good scale-down model will alsode-risk a desired process in that there will be much less likelihood ofexperiencing problems at large scale that were not previously identifiedat small scale.

Because the large bioreactors are two phase systems (gas and liquidbased) they are difficult to scale-down in a predictable manner largelydue to the solubility differences of gas types and because the fluidmechanics and/or physics of the system are very dynamic (non-linear) dueviscosity, shear, density, and surface interactions that are influencedacross scale. While bulk power dissipation rates may be similar usingtraditional bioreactor designs, the resulting bulk flow and measuredmixing times are rarely similar across all scales. As a result, problemoften arise at large scale that have not been seen as problematic in thesmall scale system because the same gradients of pH, nutrients, gasconcentration, or shear found at large scale are not easily replicatedunder a single operating state created in a small scale bioreactor.

The main design methods currently employed are geometric ratio scalingof the system (tank height to diameter ratio and an impeller to diameterratio); this being operated in combination with either 1) near constantimpeller tip speed or 2) near constant P/V (power input to volume).These approaches have been reasonably successful because it allowsoperators to compensate for some of the non-linearity of kinetics andmass transfer in a rotating agitator based system while being gassed.Whereas shear is traditionally benchmarked based upon tip speed of theimpeller (where tip speed is a function of the square of impellerdiameter). Power input to volume is more often employed because isfavorable to meeting the limits of practical mechanical designs scale-up(meaning the power requirements are within reasonable magnitude acrossscales) because power transfer is nonlinear function of speed anddiameter (and partially impeller geometry).

Once the general design of the reactor is known, common practice is tofocus on one of two critical process parameters. Choose a tier 1 targetconstant—shear sensitive cultures often use impeller tip speed, whereasmore shear tolerant cultures often use power input to volume (P/V).Mixing efficiency is important regardless of the chose scale parameter,best practice is to verify that similar or at least reasonable blendtimes are achievable across scales (T90 mixing times of <30 sec are mostdesirable, but often not achievable across scale because of bulk fluidmixing dissipation issues inherent to water based fluid). Then thesecond tier target is often tied to pH control and dissolved gases—masstransfer kLa of oxygen (primary) and kLa of carbon dioxide (secondary).Some systems are further optimized by use of computational fluid dynamic(CFD). This is a good method for determining impeller power number (Np)and for modeling the fluid flow profile in the system. Power transfercan be estimated and used to predict the amount of (mixing) powertransferred into the system. Computational or hybrid models (RPT) canalso predict fluid field direction, velocity, and localized shearconditions.

Well informed operators are often very successful in achievingacceptable outcomes if the system is designed properly, but the overallprocess is still often burdened with unknowns and time consumingtroubleshooting especially when process transfer must be facilitatedbetween dissimilar system as part of scale-up or transfer to analternate location.

The issue that is often overlooked is the bulk fluid flow of the systemand how this changes across scale. Fluid is displacement and velocity isnot linear across scales, therefore the mixing profile of liquidmovement will change not only based upon volume, tip speed, anddiameter. It also depends upon the spatial distribution of power withinthe reactor. In practical terms, designs should detune the localizedmixing performance of the smaller volume system to match the bulk mixingand mass transfer performance of the large scale system. A good designwill take in the account the effects of baffling or flow disrupters inthe design as some randomness in the mixing or vessel turn-over isbeneficial.

SUMMARY OF INVENTION

In one aspect, a scalable bioreactor system for transitioning fromtesting to commercial production is disclosed. The system may include afirst bioreactor comprising a first bioprocessing container having afirst end, a second end, and a sidewall and a first configurable mixingassembly suspended between the first and second ends of the firstbioprocessing container and a first impeller having a first diameter,the first impeller attached to the first configurable mixing assembly ina first position, wherein the first diameter and the first position areselected to achieve a set of operational parameters as well as a secondbioreactor comprising a second bioprocessing container having a firstend, a second end, and a sidewall, wherein the second bioprocessingcontainer is not the same volume as the first bioprocessing containerand a second configurable mixing assembly suspended between the firstand second ends of the second bioprocessing container and a secondimpeller having a second diameter that is not the same as the firstdiameter, the second impeller attached to the second configurable mixingassembly in a second position, wherein the second diameter and thesecond position are selected to match the set of operational parametersand the operational parameters include power per volume and impeller tipspeed. In some embodiments, the first bioprocessing container includes afirst sparger affixed to the first end of the first bioprocessingcontainer and having a first number of pores and each pore has a firstdiameter and the second bioprocessing container includes a secondsparger affixed to the first end of the second bioprocessing containerand having a second number of pores and each pore has a second diameter.In some embodiments, the first number of pores is not the same as thesecond number of pores, the first diameter is not the same as the seconddiameter, and the number of pores and pores sizes are selected so thatthe first and second bioreactors attain the same kLa. In someembodiments, the first and second locations are selected to re-entraingas bubbles rising out of the first and second spargers. In someembodiments, the first bioreactor includes a first headspace airflowdevice and the second bioreactor includes a second headspace airflowdevice and each cross-flow spargers operates to provide different ratesof airflow across a headspace to match CO2 removal rates of the liquidphase to within five percent between the first and second bioreactors.In some embodiments, the second bioreactor includes a third impellerhaving a third diameter that is not the same as the first diameter,wherein the third impeller is attached to the second configurable mixingassembly and the third diameter and third attachment location areselected in combination with the second diameter and the second positionto match the set of operational parameters. In some embodiments, theratio of the first impeller diameter to the first bioprocessingcontainer width is not the same as the ratio of the second impellerdiameter to the second bioprocessing container width. In someembodiments, the set of operational parameters further includes bulkfluid flow and T95 mixing times. In some embodiments, the set ofoperational parameters is selected based on the optimal growthconditions for a cell. In some embodiments, the cell is eukaryotic andsensitive to a shear force that increases as the impeller tip speedincreases. In some embodiments, the first bioprocessing container is abench scale volume between 0.1 liters and 50 liters and the secondbioprocessing container is a commercial volume between 50 liters and10,000 liters. In some embodiments, the first and second bioprocessingcontainers are rectangular in shape and the first and secondconfigurable mixing assemblies are offset from a center axis to increasebulk fluid flow. In some embodiments, the aspect ratio of the first andsecond bioprocessing containers is greater than 1.5. In someembodiments, the first bioprocessing container has an aspect ratiobetween 1.5 and 2 and the second bioprocessing container has an aspectratio between 1.75 and 4.

In one aspect, a method of matching fluid mixing characteristics betweenbioreactors having different volumes is disclosed. The method mayinclude selecting a first bioreactor having a set of operationalparameters, the first bioreactor comprising: a first bioprocessingcontainer having a first end, a second end, and a sidewall; and a firstconfigurable mixing assembly suspended between the first and secondsends of the first bioprocessing container The method may includeselecting a first impeller having a first diameter and attaching thefirst impeller to the configurable mixing assembly, wherein the firstdiameter and the attachment location are selected to conform to theoperational parameters. The method may include selecting a secondbioreactor, comprising: a second bioprocessing container having a firstend, a second end, and a sidewall, wherein the second bioprocessingcontainer is not the same volume as the first bioprocessing container;and a second configurable mixing assembly suspended between the firstand second ends of the second bioprocessing container. The method mayinclude selecting a second impeller having a second diameter that is notthe same as the first diameter and attaching the second impeller to thesecond configurable mixing assembly, wherein the second diameter and thesecond attachment location are selected to match the set of operationalparameters, wherein the set of operational parameters include power pervolume and impeller tip speed. The method may include the step ofselecting a third impeller having the third diameter that is not thesame as the first diameter and attaching the third impeller to thesecond configurable mixing assembly, wherein the third diameter and thethird attachment location are selected in combination with the seconddiameter and the second attachment location to match the set ofoperational parameters. In some embodiments, the step of adding thethird impeller reduces the required second and third impeller tip speedsto maintain a power per volume and impeller tip speed in the first andsecond bioreactors. In some embodiments, the ratio of the first impellerdiameter to the first bioprocessing container width is not the same asthe ratio of the second impeller diameter to the second bioprocessingcontainer width. In some embodiments, the set of operational parametersfurther includes bulk fluid flow and T95 mixing times. In someembodiments, the set of operational parameters is selected based onoptimal growth conditions for a cell. In some embodiments, the cell iseukaryotic and sensitive to a shear that increases as the impeller tipspeed increases. In some embodiments, the first bioprocessing containeris a bench scale volume between 0.1 liters and 50 liters and the secondbioprocessing container is a commercial volume between 50 liters and10,000 liters. In some embodiments, the first and second bioprocessingcontainers are rectangular in shape and the first and secondconfigurable mixing assemblies are offset from a center axis to increasebulk fluid flow. In some embodiments, the aspect ratio of the first andsecond bioprocessing containers is greater than 1.5.

In one aspect, a method of matching fluid mixing characteristics betweenbioreactors having different volumes is disclosed. The method mayinclude selecting a first bioreactor having an operational parameter,the first bioreactor comprising a first bioprocessing container having afirst end, a second end, and a sidewall and a first configurable mixingassembly suspended between the first and seconds ends of the firstbioprocessing container. The method may include the step of selecting afirst sparger having a first number of pores, wherein the pores have afirst diameter, wherein in the first number and first diameter areselected to conform to the operational parameter, wherein the firstsparger is affixed to the first end. The method may include the step ofselecting a second bioreactor, comprising a second bioprocessingcontainer having a first end, a second end, and a sidewall, wherein theaspect ratio of the second bioprocessing container is not the same asthe aspect ratio of the first bioprocessing container and a secondconfigurable mixing assembly suspended between the first and second endsof the second bioprocessing container. The method may include the stepof selecting a second sparger having a second number of pores, whereinthe pores have a second diameter and the second sparger is affixed tothe first end of the second bioprocessing container, wherein the secondnumber of pores and first number of pores are not the same and thesecond diameter and first diameter are not the same, wherein the secondnumber of pores and second diameter are selected to match theoperational parameter to within five percent, wherein the operationalparameter is kLa. In some embodiments, the first bioreactor includes afirst headspace airflow device and the second bioreactor includes asecond headspace airflow device and each headspace airflow deviceoperates to provide different rates of airflow across a headspace tomatch the operational parameter. The method may include the stepattaching a second impeller to the second mixing assembly, wherein thesecond impeller is configured to re-entrain gas bubbles rising out ofthe second sparger, wherein the location of the sparger and secondimpeller in combination with the second number and second pore size areselected to match the operational parameter. In some embodiments, theaspect ratio of the first bioprocessing container is between 1.5 and 2and the aspect ratio of the second bioprocessing container is between1.75 and 4. In some embodiments, the first bioprocessing container is abench volume between 0.1 liters and 50 liters and the secondbioprocessing container is a commercial volume between 50 liters and10,000 liters. In some embodiments, the first and second bioprocessingcontainers are rectangular in shape and the first and second mixingassemblies are offset from a center axis to achieve a desired kLa.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

To easily identify the discussion of any particular element or act, themost significant digit or digits in a reference number refer to thefigure number in which that element is first introduced.

FIG. 1 illustrates a mixing system 100 in accordance with oneembodiment.

FIG. 2 illustrates a mixing system 200 in accordance with oneembodiment.

FIG. 3 illustrates a flexible compartment 300 in accordance with oneembodiment.

FIG. 4 illustrates a mixing system 400 including a flexible compartment402 and helical assembly 426 in accordance with one embodiment.

FIG. 5 illustrates an exploded view of a helical assembly 500 inaccordance with one embodiment.

FIG. 6 illustrates a mixing system 600 including a flexible container604 and sparger in accordance with one embodiment.

FIG. 7 illustrates a sparger 700 design in accordance with oneembodiment.

FIG. 8 illustrates a sparger 800 in accordance with one embodiment.

FIG. 9 illustrates a mixing system 900 including a flexible compartment902 and offset drive shaft 910 in accordance with one embodiment.

FIG. 10 illustrates a mixing system 1000 including a flexiblecompartment 902 having an offset and angled drive shaft 910 inaccordance with one embodiment.

FIG. 11 illustrates a mixing system 1100 including a gas delivery systemin accordance with one embodiment.

FIG. 12 illustrates a gas delivery system 1200 in accordance with oneembodiment.

FIG. 13 illustrates a mixing system 1300 showing gas bubble trajectoryaccording a single impeller embodiment.

FIG. 14 illustrates a mixing system 1400 showing gas bubble trajectoryhaving a suboptimally placed sparger.

FIG. 15 illustrates a mixing system 1500 showing gas bubble trajectoryhaving a suboptimally placed second impeller.

FIG. 16 illustrates a mixing system 1600 showing gas bubble trajectoryhaving an optimally placed sparger and set of impellers.

FIG. 17 illustrates a sparger layout 1700 in accordance with oneembodiment.

FIGS. 18A-F illustrate a gas distribution pattern using differentsparger locations in accordance with one embodiment.

FIG. 19 illustrates gas bubble residence times 1900 from gas arising outof various sparger locations in accordance with one embodiment.

FIG. 20 illustrates a kLa trend 2000 using various sparger locations inaccordance with one embodiment.

FIG. 21 illustrates a sparger performance 2100 in accordance a varietyembodiments.

FIG. 22 illustrates a mixing consistency 2200 across volumetric scales.

FIG. 23 illustrates a matching operational parameters chart 2300 inaccordance with one embodiment.

FIG. 24 illustrates a method of matching fluid mixing characteristicsbetween bioreactors having different volumes.

FIG. 25 illustrates a method of match fluid mixing characteristicsbetween bioreactors having different volumes.

FIG. 26 illustrates Kscore comparisons between legacy bioreactors havingdifferent volumes.

FIG. 27 illustrates kLa comparison data across scales.

FIG. 28 illustrates projected Kscores between scales in the systemdescribed herein.

DETAILED DESCRIPTION Description

Embodiments of systems, methods, and apparatuses for bioreactor scalingare described in the accompanying description and figures. In thefigures, numerous specific details are set forth to provide a thoroughunderstanding of certain embodiments. A skilled artisan will be able toappreciate that the scalable bioreactor systems and methods describedherein may be used for a variety of applications including, but notlimited to, bringing a cell culture product from laboratory or benchscale to commercial scale production. Additionally, the skilled artisanwill appreciate that certain embodiments may be practiced without thesespecific details. Furthermore, one skilled in the art will readilyappreciate that the specific sequences in which methods are presentedand performed are illustrative and it is contemplated that the sequencesmay be varied and still remain within the spirit and scope of certainembodiments.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Furthermore, in described various embodiments, the specification mayhave presented a method and/or process as a particular sequence ofsteps. However, to the extent that the method or process does not relyon the particular order of steps set forth herein, the method or processshould not be limited to the particular sequence of steps described. Asone of ordinary skill in the art would appreciate, other sequences ofsteps may be possible. Therefore, the particular order of the steps setforth in the specification should not be construed as limitations on theclaims. In addition, the claims directed to the method and/or processshould not be limited to the performance of their steps in the orderwritten, and one skilled in the art will readily appreciate that thesequence may be varied and still remain within the spirit and scope ofthe various embodiments.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

The invention described herein is a scalable bioreactor system and setof methods that enables manufacturers in the bioproduction space topredict the physiological growth conditions previous discussed byaltering the hardware between scales to achieve similar mixing profilesand growth conditions between the two or more scales. For example, ascaled bioreactor system may include two or more different sizedbioreactors that achieve similar bulk fluid, similar shear forces at theimpeller tip, and similar power input per volume ratios by altering thegeometry of the tanks, size and number of impellers, impeller to tankdiameter (width) ratios, sparge designs, and CO2 blanket removal systemsbetween the two or more scales. Stated differently, the two or morebioreactors in the present invention can have vastly different physicalcharacteristics while still achieving similar mixing profiles and growthconditions in order to make predictions for commercial scalebioproduction systems at small scales, including, benchtop scales.

The magnitude of localized shear, mixing power dissipation rate, andbulk fluid flow should all be more fully addressed in scale-up design.This can be done by optimizing the power distribution of impellers(increasing the quantity of impeller in larger vessels). By putting alimit of how far impeller are spaced (increasing quantity as volumeincreases) or optimizing bulk fluid mixing properties using CFD or RPTmodels and also altering the size or shape of the impellers (notnecessary the shape but either the swept area, blade geometry, surfacetexture properties, or diameter in order to match hydrodynamic (eddy)profiles created by fluid movement of the agitator). The primary goal isto achieve nearly identical maximum shear levels across at least 3magnitudes of volume scale while delivering similar P/V, bulk flow, T95mixing times, and doing so with a reasonable power input values for cellculture and within practical limits imposed by cell culture sensitivitythat can occur in very large working volumes (>1000 L). The inventiondescribed herein will enable reactor scale-up to be more predictableacross scale and thus more easily/logically tuned because the sheardistribution is similar and the bulk fluid movement is nearly identical.The objective of the current invention is to address the unmet need thatnow exists due to a wide breadth of cell culture process sensitivityvariances that are inherent impacted when altering cell lines, fluidtype, cell density, or media formulations.

The methods herein, are designed to achieve the desired outcome ofdesigning and characterizing reactor scale-up will be more predictableacross scale and thus more easily/logically tuned because the sheardistribution is more similar and the bulk fluid movement is nearlyidentical. The objective here is to address the unmet need that nowexists due to a wide breadth of cell culture process sensitivityvariances that are inherent impacted when altering cell lines, fluidtype, cell density, or media formulations.

In regards to non-circular vessels, using rectangular vessels with acenter drive agitator that is off set from center line is beneficial formany reasons. The unbalanced design of these two geometries (non-squareand non-centered) may create improved baffling effects and desirablemixing turn over (bulk flow) within the system. Our data supports theidea that mass transfer and mixing performance is improved through ourperformance measurement are showing >2× improvement in kLa and mixingcompared to circular designs at similar working volumes. The scaling ofthe impeller to match tip speed and P/V will result in a proportionallysmaller impeller as the vessel rated working volumes decreases. Thishelps to compensate for the inherent changes in fluid mechanics thatoccur at smaller scale (proportionally larger impeller to vessel wallclearance will encourage and induce bulk flow much more representativeto that of large scale systems). These mixing design features are thencombined with optimal sparge position and this will be responsible fordramatically increasing bioreactor efficiency at large scale. Two phasemixing efficiency gains will increase bubble residence time within theliquid column and overall improved bubble distribution from the multipleimpellers are both highly desirable attributes that are known tosignificantly improve mass transfer performance.

FIG. 1 illustrates a fluid mixing system 100 according to variousembodiments. The mixing system 100 generally comprises a rigid housing102, a motor 104 mounted to the rigid housing 102, a first bearingassembly 106 in rotational communication with the motor 104 through adrive shaft 120 and providing rotational movement to the interior of aflexible compartment 118, a hinges 108 to secure a door 110 to the rigidhousing 102 and provide enclosure for the flexible compartment 118, arigid housing support 112 for the rigid housing 102 to mount thereto,and a support wheels 114 affixed to the rigid housing support 112 andprovide mobility to the mixing. The rigid housing 102 may have rigidhousing openings 122 cut into rigid housing floor 124 for retaining oneport 228 or more and a second bearing assembly 222 from the flexiblecompartment 218. In some embodiments, the rigid housing may be fixed inplace and not require support wheels 114. In such embodiments, the rigidhousing support 112 may be bolted to the bolt or simply held in place bythe weight of the rigid housing 102.

FIG. 2 illustrates a cross sectional view of a fluid mixing system 200according to various embodiments. The mixing system 200 comprises amotor 202 mounted to a rigid housing 208 having a drive shaft 120 thatis in sterile, rotational communication to the interior of a flexiblecompartment 218 through a first bearing assembly 204. The mixing system200 also comprises a helical assembly 214 comprised of a yoke 230 and ayoke/impeller 232 that act to suspend a driveline 206 between a firstend 234 and second end 236 of the flexible compartment 218. Theyoke/impeller 232 may be mounted to a second bearing assembly 222 toprovide rotational movement to the helical assembly 214 on an opposingend of the flexible compartment 218. One impeller 216 or more may bemounted to the helical assembly 214 to provide mixing to a fluid withinthe flexible compartment 218. To facilitate installation of the flexiblecompartment 218 into the rigid housing 208 a pull handle 220 may bemounted to the second end 236 of the flexible compartment 218 and insome embodiments onto the second bearing assembly 222. The rigid housing208 may be mounted to a rigid housing support 224 and support wheels 226may be attached to the rigid housing support 224 to provide mobility tothe mixing system 200. In various embodiments, the flexible compartment218 further comprises at least one port 228 that may protrude throughthe rigid housing floor 124, 238.

In various embodiments, a user can open the door 110 to the rigidhousing 102, 208 for easy installation of the flexible compartment 118,218. As seen in FIG. 1, when the door 110 move to an open position thetop surface 126 of the rigid housing 102, 208 may be completely open onthe front face. The top surface 126 may make a “U” perimeter shape thatcomprises a back portion and two side portions that extend toward thedoor. While the door 110 is in the open configuration the flexiblecompartment 118, 218 may be moved into the chamber of the rigid housing102, 208. The first bearing assembly 106, 204 located on the first end116, 234 of the flexible compartment 118, 218 may then be inserted ontothe drive shaft 120, 240. Additional disclosure relating to mounting theflexible compartment 118, 218 to the drive shaft 120 may be found in US2017-0183617, filed on Dec. 28 of 2016 which is incorporated herein byspecific reference in its entirety. Hangers (not shown) attached to therigid housing 102, 208 may be hooked onto loops (not shown) on theflexible compartment 118, 218 to further secure the flexible compartment118, 218 to the top surface 126 of the rigid housing 102, 208. Once thefirst end 116 of the flexible compartment 118, 218 is secured to the topsurface 126 of the rigid housing 102, 208 the second end 236 may slideinto the rigid housing floor 124, 238. In various embodiments, theflexible compartment 118, 218 will comprise one port 228 or more and asecond bearing assembly 222 that protrude from the exterior of thesecond end 236 of the flexible compartment 118, 218. Rigid housingopenings 122 in the rigid housing floor 124, 238 may be configured toaccept the one port 228 or more and second bearing assembly 222,thereby, securing the second end 236 of the flexible compartment 118,218 to the rigid housing floor 124, 238 of the rigid housing 102, 208.In some embodiments, a closure (not shown) can cover the rigid housingopenings 122 to further secure the one port 228 or more and secondbearing assembly 222 to the rigid housing floor 124, 238 of the rigidhousing 102, 208. In various embodiments, a user can grip the pullhandle 220 located at the second end 236 of the flexible compartment118, 218 to pull the flexible compartment 118, 218 into place within therigid housing 102, 208.

In various embodiments, once installation has been accomplished a fluidmay be fed into the sterile flexible compartment 118, 218 which mayrequire mixing. The motor 104, 202 may be activated using a controller(not shown) which may then rotate the drive shaft 120, 240 which wasinserted previously into the first bearing assembly 106. In someembodiments, there may be a single drive shaft 120, 240 that protrudesfrom the motor 104, 202 and into the sterile flexible compartment 118,218 and in other embodiments the first bearing assembly 106 will beclosed off and have a second drive shaft portion 242 that extends fromthe first bearing assembly 106. In various embodiments, the drive shaft120 or second drive shaft portion 242 will mount to a yoke 230 thatworks to space apart a first line 210 and a second line 212 of adriveline 206. On the second end 236 of the flexible compartment 118,218 there may be a second bearing assembly 222 comprising ayoke/impeller 232 that operates to suspend the other ends of the firstline 210 and the second line 212 as well as provide mixing as itrotates. The second bearing assembly 222 may be designed to providerotational movement so that rotational to allow the helical assembly 214to freely rotate as the motor 104, 202 drives the helical assembly 214from the opposing end. One impeller 216 or more may provide mixing inaddition to the yoke/impeller 232.

In various embodiments, an added advantage of the yoke/impeller 232 isto provide very low volume mixing. For example, a bioreaction mayrequire a small volume at the beginning of a reaction and the fluidvolume may be increased as the bioreaction matures. Currently availablebioreactors have limitations with scale-up which the present embodimentreduces. One impeller 216 or more may be affixed at various locations onthe helical assembly 214 when considering optimal scale up for a givenbioreactor as well. In some embodiments, the yoke/impeller 232 maymaintain a homogenous mix in the fluid at very low volume during adraining process.

FIG. 3 illustrates a flexible compartment 300 according to variousembodiments. The flexible compartment 300 comprises a first end 302, anopposing second end 304, a sidewall 306 connecting the first end 302 andthe second end 304, at least three panels 308 joining the first end 302and the second end 304, a sidewall line 310, a centerline 312, and acornerline 314.

In various embodiments, the centerline 312 is an indicator of a verticalaxis running from the center of the first end 302 to the center of thesecond end 304 of the flexible compartment 300. For example, thecenterline 312 may be placed such that the length from the centerline312 to opposing panels 308 is equal. In various embodiments, a sidewallline 310 may be an indicator of a plane running from the first end 234to the second end 304 of the flexible compartment 300 and extend fromthe centerline 312 to the mid-point of a panel 308. In variousembodiments, a cornerline 314 may be an indicator of a plane runningform the first end 302 to the second end 304 of the flexible compartment300 and extend from the centerline 312 to where two panels 308 arejoined to form a corner. In various embodiments, the indicators listedabove may be used to determine where the helical assembly 214 willreside within the flexible compartment 300 when reducing dead zones andincreasing bulk fluid and, thereby, increasing overall mixing efficiencywithin the mixing system 100, 200.

FIG. 4 illustrates a mixing system 400 according to various embodiments.The mixing system 400 comprises a flexible compartment 402 having afirst end 404 and an opposing second end 406 that are joined together bya sidewall 408 having at least three panels 410 and sidewall corners 412where the panels meet. The flexible compartment 402 may further includeone or more inlets 414, one or more outlets 416, one or more spargers418, one or more sensor ports 420 that optionally contain a sensor 422,and a drain 424. In various embodiments a helical assembly 426 may besuspended between the first end 434 and the second end 436 of theflexible compartment 402 and have one or more impellers 428 positionedthereto. In various embodiments, a drive shaft 430 may project into afirst bearing assembly 432 and the first bearing assembly 432 mayprovide a sterile connection between the drive shaft 430 on the exteriorof the flexible compartment 402 to a yoke on the interior of theflexible compartment 402. In various embodiments, a second bearingassembly 440 may be positioned on the second end 406 of the flexiblecompartment 402 and may include a pull handle 444 projecting onto theexterior portion of the flexible compartment 402 and on theopposing/interior portion the second bearing assembly 440 may connect toa yoke/impeller 442. In various embodiments, the helical assembly 426may be comprised of a first line 446 and a second line 448 that eachhave a first end 434 connected to a yoke 438 and a second end 436connected to a yoke/impeller 442 and during operation the rotationalmovement may be applied to mix a fluid 450 within the flexiblecompartment 402. In various embodiments, the flexible compartment 402may include an attachment ring 452 either affixed to or molded as partof the second bearing assembly 222 used can slide into a retentiondevice on the rigid housing 102, 208 during installation. In someembodiments, the design may include a snap ring that fits onto a pin andmay slide into the bottom port of the flexible compartment 402.

In various embodiments, the flexible compartment 402 may include one ormore inlets 414 and outlets 416. Inlets 414 may be used during theinstallation process to add a gas to the flexible compartment 402 inorder to inflate the flexible compartment 402 to its working volume.Additionally, inlets 414 may be used to introduce dry media, buffers,liquid nutrients, or anything else requiring mixing. An outlet 416 maybe used to harvest the contents of the flexible compartment 402 after amixing process is complete or a bioreaction has achieved a desiredstate. Additionally, a drain 424 may be used to empty the waste withinthe flexible compartment 402. There are various ways known in the artfor attaching inlets 414, outlets 416, and drains 424. A commontechnique is weld the component to the flexible compartment 402. Forexample the component may include a polymer that can be welded to thepolymer comprising the flexible compartment 402. US 2017-0183617includes a list of common weldable materials used to produce flexiblecompartments 402.

In various embodiments, sensors 422 may be used to monitor theenvironmental conditions within the flexible compartment 402. There area variety of sensors and sensor ports 420 available on the marketincluding those described in US 2008-0032389 filed on Mar. 26 of 2007which is incorporated herein by specific reference in its entirety.Various techniques are described in the above cited reference disclosingways to bond sensor ports 420 to flexible compartments 402 using weldingand adhesion methods.

In various embodiments, the mixing system 400 described herein may beused for cultivating cells and then harvesting the cells in theirentirety or harvesting a cell byproduct such as a protein or enzyme.Such bioreactions often require introduction of a gas which is typicallydone with using a sparger 418 in the field of bioproduction. A varietyof sparger 418 designs and their methods of attachment are described inUS 2013-0082410 filed on Sep. 28 of 2012 which is incorporated herein byspecific reference in its entirety.

In various embodiments, the first bearing assembly 432 and the secondbearing assembly 440 may include a first annular sealing flange 454 anda second annular sealing flange 456 that may be sealed to openings onthe flexible compartment 402 by welding or adhesive around theperimeter. As disclosed in US 2017-0183617 this allows for rotationalmovement of a hub while an outer casing remains fixed to the flexiblecompartment 402 allowing the helical assembly 426 to freely rotatewithin the flexible compartment 402 while remaining sterile to theexterior.

In various embodiments, an attachment ring 452 may be engageable to aretention device on the rigid housing 102, 208. The retention device maytake the form of a bracket or some other physical structure capable ofretaining and/or restricting the movement of the attachment ring 452.Generally, during the installation process a user will pull the pullhandle 444 into the rigid housing opening 122 in order to facilitate theattachment ring 452 and retention device interaction in order tocomplete flexible compartment 402 installation.

In various embodiments, the optimal location of the helical assembly 426relative to the flexible compartment 402 will be along the centerline312 as depicted in FIG. 4.

FIG. 5 is an illustration of an exploded view of a portion of a helicalassembly 500 according to various embodiments. The helical assembly 500may comprise a first line 502, a second line 504, one or more rungs 516,one or more stabilizers 524, and one or more impellers 536. Each of thelines may include a first end 506, 510 and a second end 508, 512.

In various embodiments, the helical assembly may comprise one or morerungs 516 having a first protrusion 518 that projects through an opening514 on a first line 502 and a second protrusion 520 that projectsthrough an opening 514 on a second line 504. In some embodiments, rungcaps 522 may snap onto the protrusions 518, 520 to secure the rungs 516to the lines 502, 504.

In various embodiments, the helical assembly 500 may include astabilizer 524 that includes a crossmember 526 having a first end 530that projects through an opening 514 on the first line 502 and a secondend 532 that projects through an opening 514 on the second line 504.Stabilizer caps 534 may snap onto the ends 530, 532 to secure thestabilizer 524 onto the helical assembly 500. In some embodiments, astem 528 may project from the center and perpendicular to thecrossmember 526.

In various embodiments, an impeller 536 may include fins 538 having afirst attachment 542 that projects through an opening 514 on the firstline 502 and a second attachment 544 that projects through an opening514 on the second line 504. In some embodiments impeller caps 546 maysnap onto the attachments 542, 544 to secure the impeller 536 onto thehelical assembly 500. In various embodiments, a receiver 540 may extendfrom the center and perpendicular to the impeller 536.

In various embodiments, the receiver 540 may be tubular in nature andaccept a stem 528 from the stabilizer 524. The receiver 540 and stem 528may slide relative to one another as the rotational rate of the helicalassembly 500 varies.

FIG. 6 is an illustration of one embodiment of a mixing system 600incorporating features of the present invention. The mixing system 600comprises a substantially rigid support rigid support housing 602 inwhich a flexible container 604 is disposed. Rigid support housing 602has an upper end 606, a lower end 608, and an interior surface 666 thatbounds a compartment 612. Formed at lower end 608 is a floor 614. Anencircling sidewall 616 extends up from floor 614 toward upper end 606.As will be discussed below in greater detail, one opening 618 or morecan extend through floor 614 or sidewall 616 of rigid support housing602 so as to communicate with compartment 612. Examples of spargerdevices and systems that can be used in the presently disclosedinvention are disclosed in U.S. Pat. No. 9,643,133 that issued on May 9,2017 which is hereby incorporated by specific reference.

Upper end 606 terminates at a lip 620 that bounds an inlet opening 622to compartment 612. If desired, a cover, not shown, can be mounted onupper end 606 so as to cover inlet opening 622. Likewise, an accessopening can be formed at another location on rigid support housing 602such as through sidewall 616 at second end or through floor 614. Theaccess opening is large enough so that an operator can reach through theaccess opening to help manipulate and position flexible container 604.The access opening can be selectively closed by a door or cover plate.

It is appreciated that rigid support housing 602 can come in a varietyof different sizes, shapes, and configurations. For example, floor 614can be flat, frustoconical, or have other slopes. Sidewall 616 can havea transverse cross section that is circular, polygonal or have otherconfigurations. Rigid support housing 602 can be insulated and/orjacketed so that a heated or cooled fluid can flow through the jacketfor heating or cooling the fluid contained within flexible container604. Flexible container 604 can be any desired volume such as thosediscussed below.

As also depicted in FIG. 6, flexible container 604 is at least partiallydisposed within compartment 612 of support rigid support housing 602.Flexible container 604 comprises a container 624 having one or moreports 626 mounted thereon. In the embodiment depicted, container 624comprises a flexible bag having an interior surface 610 that bounds achamber 628 suitable for holding a fluid 630 or other type of material.More specifically, container 624 comprises a side wall 632 that, whencontainer 624 is inflated, can have a substantially circular orpolygonal transverse cross section that extends between a first end 634and an opposing second end 636. First end 634 terminates at a top endwall 638 while second end 636 terminates at a bottom end wall 640.

Container 624 can be comprised of one or more sheets of a flexible,water impermeable material such as a low-density polyethylene or otherpolymeric sheets having a thickness typically in a range between about0.1 mm to about 5 mm with about 0.2 mm to about 2 mm being more common.Other thicknesses can also be used. The material can be comprised of asingle ply material or can comprise two or more layers that are eithersealed together or separated to form a double wall container. Where thelayers are sealed together, the material can comprise a laminated orextruded material. The laminated material can comprise two or moreseparately formed layers that are subsequently secured together by anadhesive.

The extruded material can comprise a single integral sheet thatcomprises two or more layers of different material that are eachseparated by a contact layer. All of the layers are simultaneouslyco-extruded. One example of an extruded material that can be used in thepresent invention is the HyQ CX3-9 film available from HyCloneLaboratories, Inc. out of Logan, Utah. The HyQ CX3-9 film is athree-layer, 9 mil cast film produced in a cGMP facility. The outerlayer is a polyester elastomer coextruded with an ultra-low densitypolyethylene product contact layer. Another example of an extrudedmaterial that can be used in the present invention is the HyQ CX5-14cast film also available from HyClone Laboratories, Inc. The HyQ CX5-14cast film comprises a polyester elastomer outer layer, an ultra-lowdensity polyethylene contact layer, and an EVOH barrier layer disposedtherebetween. In still another example, a multi-web film produced fromthree independent webs of blown film can be used. The two inner webs areeach a 4 mil monolayer polyethylene film (which is referred to byHyClone as the HyQ BM1 film) while the outer barrier web is a 5.5 milthick 6-layer co-extrusion film (which is referred to by HyClone as theHyQ BX6 film).

The material can be approved for direct contact with living cells and becapable of maintaining a solution sterile. In such an embodiment, thematerial can also be sterilizable such as by ionizing radiation.Examples of materials that can be used in different situations aredisclosed in U.S. Pat. No. 6,083,587 that issued on Jul. 4, 2000 andU.S. Patent Publication No. US 2003/0077466 A1, published Apr. 24, 2003that are each hereby incorporated by specific reference.

In one embodiment, container 624 comprises a two-dimensional pillowstyle bag wherein two sheets of material are placed in overlappingrelation and the two sheets are bounded together at their peripheries toform internal chamber 628. Alternatively, a single sheet of material canbe folded over and seamed around the periphery to form internal chamber628. In another embodiment, container 624 can be formed from acontinuous tubular extrusion of polymeric material that is cut to lengthand the ends seamed closed.

In still other embodiments, container 624 can comprise athree-dimensional bag that not only has an annular side wall but also atwo-dimensional top end wall 638 and a two dimensional bottom end wall640. For example, three-dimensional container 624 can comprise sidewall616 formed from a continuous tubular extrusion of polymeric materialthat is cut to length, such as shown in FIG. 7. A circular top end topend wall 638 and bottom end wall 640 can then be welded to opposing endsof sidewall 616. In yet another embodiment, three-dimensional container624 can be comprised of a plurality of discrete panels, typically threeor more, and more commonly between four to six. Each panel can besubstantially identical and comprises a portion of side wall 632, topend wall 638, and bottom end wall 640 of container 624. The perimeteredges of adjacent panels are seamed together to form container 624. Theseams are typically formed using methods known in the art such as heatenergies, RF energies, sonics, or other sealing energies. In alternativeembodiments, the panels can be formed in a variety of differentpatterns.

It is appreciated that container 624 can be manufactured to havevirtually any desired size, shape, and configuration. For example,container 32 can be formed having chamber 628 sized to 10 liters, 30liters, 100 liters, 250 liters, 500 liters, 750 liters, 1,000 liters,1,500 liters, 3,000 liters, 5,000 liters, 10,000 liters or other desiredvolumes. Chamber 628 can also have a volume in a range between about 10liters to about 5,000 liters or about 30 liters to about 1,000 liters.Any other ranges selected from the above volumes can also be used.Although container 624 can be any shape, in one embodiment container 624is specifically configured to be complementary to or substantiallycomplementary to compartment 612 of rigid support housing 602.

In any embodiment, however, it is typically desirable that whencontainer 624 is received within compartment 612, container 624 isgenerally uniformly supported by support rigid support housing 602.Having at least generally uniform support of container 624 by rigidsupport housing 602 helps to preclude failure of container 624 byhydraulic forces applied to container 624 when filled with fluid.

Although in the above discussed embodiment container 624 is in the formof a flexible container 604, in alternative embodiments it isappreciated that container 624 can comprise any form of collapsiblecontainer, flexible container 604, or semi rigid container. Furthermore,in contrast to having a closed top end wall 638, container 624 cancomprise an open top liner. Container 624 can also be transparent oropaque and can have ultraviolet light inhibitors incorporated therein.

Mounted on top end wall 638 are a plurality of ports 626 that are influid communication with chamber 628. Although two ports 626 are shown,it is appreciated that one or three or more ports 626 can be presentdepending on the intended use of container 624. As such, each port 626can serve a different purpose depending on the type processing to beundertaken. For example, ports 626 can be coupled with a tube 642 fordispensing fluid or other components into chamber 628 or withdrawingfluid from chamber 628. In addition, such as when container 624 is usedas a bioreactor for growing cells or microorganisms, ports 626 can beused to provide various probes, such as temperature probes, pH probes,dissolved oxygen probes, and the like, access to chamber 628. It isappreciated that ports 626 can come in a variety of differentconfigurations and can be placed at any number of different locations oncontainer 624, including sidewall 616 and bottom end wall 640.

Although not required, in one embodiment means are provided for mixingfluid 630 within chamber 628. The means for mixing can be in the form ofa mixing assembly. By way of example and not by limitation, in oneembodiment as shown in FIG. 6 a drive shaft 646 projects into chamber628 and has an impeller 648 mounted on the end thereof. A dynamic seal650 forms a seal between drive shaft 646 and container 624. Externalrotation of drive shaft 646 facilitates rotation of impeller 648 thatmixes and/or suspends fluid 630 within a chamber 628. Specific examplesof how to incorporate a rotational mixing assembly into a flexiblecontainer are disclosed in U.S. Pat. No. 7,384,783 that issued Jun. 10,2008 and U.S. Pat. No. 7,682,067 that issued on Mar. 23, 2010, which areincorporated herein by specific reference.

In yet another alternative embodiment of the means for mixing or themixing assembly, mixing can be accomplished by vertically reciprocallymoving a vertical mixer within chamber 628. Further disclosure withregard to the assembly and operation of vertical mixer is disclosed inU.S. Patent Publication No. 2006/0196501, published Sep. 7, 2006, whichis incorporated herein by specific reference. In yet other embodiments,it is appreciated that the mixing can be accomplished by simplycirculating fluid through chamber 628 such as by using a peristalticpump to move fluid in and out of chamber 628 by rotating a magneticimpeller or stir bar within container 624 and/or by injecting sufficientgas bubbles within the fluid to mix the fluid. Other conventional mixingtechniques can also be used.

Continuing with FIG. 6, bottom end wall 640 has a plurality of spargersincorporated therein. Specifically, bottom end wall 640 comprises afirst sheet 652 having a first side face 654 and an opposing second sideface 656. The first sheet 652 overlays a second sheet 658 that likewisehas a first side face 660 and an opposing second side face 662. Firstsheet 652 and second sheet 658 typically comprise flexible polymericsheets such as those discussed above with regard to container 624. Asdiscussed above with regard to bottom end wall 640, the first sheet 652can comprise a continuous sheet that is welded to the side wall 632around a perimeter edge 702 as depicted in FIG. 7. Alternatively, firstsheet 652 can comprise an integral portion of sidewall 616 or cancomprise a plurality of separate sheets secured together that are eitherattached to or are an integral portion of sidewall 616. Second sheet 658can be welded to second side face 656 of first sheet 652 and/or weldedto sidewall 616, such as along a perimeter edge 704 of the second sheet658. In other embodiments, second sheet 658 can be welded to or comprisean integral portion of sidewall 616, as discussed above with regard tofirst sheet 652, while the first sheet 652 is welded or otherwisesecured to first side face 660 of second sheet 658 and/or sidewall 616.

Depicted in FIG. 8 is a top plan view of first sheet 652 overlayingsecond sheet 658. In this embodiment, the first sheet 652 and the secondsheet 658 are welded together by a weld line 802. Weld line 802, as withother weld lines discussed herein, can be formed using any conventionaltechnique such as laser welding, sonic welding, heat welding, or thelike. Weld line 802 is shown as welding together the perimeter oroutside edges of the first sheet 652 and the second sheet 658 but can beformed radially inward from one or both of the perimeter edges or atother locations. As also shown in FIG. 8, four separate spargers 804,806, 808, 810 are formed by producing other weld lines between the firstsheet 652 and the second sheet 658.

For example, spargers 804, 806, 808, 810 are formed by forming weldlines 812, 814, 816, 818 starting at first locations 820 located at oradjacent to the perimeter edge of first sheet 652 and/or second sheet658 and extending into the interior of the first sheet 652 and thesecond sheet 658 along a predetermined path for the sparger 804 and thencircles back to a second location 822 at or adjacent to the perimeteredge of the first sheet 652 and/or the second sheet 658 adjacent tofirst location 820. Weld line 812 bounds a perimeter of a spargerpathway 824 which is the area bounded between the first sheet 652 andthe second sheet 658 and partially encircled by weld line 812. In theembodiment depicted, sparger pathway 824 comprises a gas transfer path826 that extends from a first end 830 to an opposing second end 834. Anopening 828 is formed at first end 830 between the first location 820and the second location 822 and between the first sheet 652 and thesecond sheet 658 through which a gas can be fed into gas transfer path826. Sparger pathway 824 also comprises a sparging area 832 formed atsecond end 834 that is in fluid communication with gas transfer path826. In the embodiment depicted, gas transfer path 826 is a narrowelongated path while sparging area 832 forms an enlarged circular area.Other configurations can also be used.

A plurality of perforations 836 extend through first sheet 652 ofsparging area 832 so that gas can pass along gas transfer path 826, intosparging area 832 and then out through perforations 836 to form gasbubbles within fluid 630 disposed within chamber 628. Spargers 806, 808,810 are similarly formed with like reference characters being used toidentify like elements. By using this technique, a plurality of discretespargers can be easily formed on container 624. Each sparger can bedisposed at any desired location and be any desired size, shape orconfiguration. Likewise, although four spargers are shown, it isappreciated that any number of spargers such as 1, 2, 3, 5, or more canbe formed with the first sheet 652 and the second sheet 658. Thesparging areas can be uniformly distributed over the first sheet 652 andthe second sheet 658 or can be located at defined locations for optimalsparging. For example, a sparger can be disposed directly below themeans for mixing such that the mixing or movement of fluid 630 producedby the mixer helps to entrain the gas bubbles within fluid 630.

In some embodiments, each sparger can have the same number ofperforations 836 and all perforations 836 can be the same size andshape. In alternative embodiments, perforations 836 can be differentbetween two or more different spargers. For example, different spargerscan have different numbers, sizes, and/or shapes of perforations 836 tooptimize performance in different situations. Larger perforations 836produce larger gas bubbles that may be optimal for stripping CO2 fromfluid 630 whereas smaller perforations produce smaller bubbles that maybe preferred for oxygenating fluid 630. Likewise, increasing the numberof perforations 836 may be helpful in causing the bubbles to mix thefluid and/or increase stripping or oxygenation. In other embodiments, itis appreciated that one or more of spargers 804, 806, 808, 810 can havecombinations of different perforations 836. For example, a singlesparger can have both small and large perforations 836. In oneembodiment, the smaller bubbles are formed from perforations 836typically having a diameter of less than 0.8 mm, 0.4 mm or 0.2 mm, 0.1mm while the large bubbles are formed from perforation typically havinga diameter greater than 1.5 mm, 0.8 mm, 0.4 mm or 0.15 mm. Perforationsof other diameters can also be used. The size of the perforation andresulting bubbles depends on the intended use and the size of container624. For example, the large bubbles are typically larger when processinga large volume of fluid in a large container than when processing arelatively small volume of fluid in a small container. The variance ordelta between the diameter of the perforations for the small bubbles andthe perforations for the large bubbles is typically at least 0.15 mm,0.3 mm, 0.5 mm or 1 mm and is often within ±0.1 mm or ±0.5 of thesevalues. Other variances can also be used.

As discussed below in greater detail, spargers 804, 806, 808, 810 cansimultaneously operate or, alternatively, a manifold or other regulatorcan be used so that one or more of the spargers can be operated whilethe other spargers are not operated. Accordingly, by having differentspargers with different perforations 836, select spargers can be used indifferent situations or times to optimize performance.

In some embodiments, it is appreciated that gas transfer gas transferpath 826 of sparger 804 is not required. For example, perforations 836can be formed through first sheet 652 overlying gas transfer path 826 soas to convert gas transfer path 826 in a portion of sparging area 832.It is appreciated that perforations 836 can be formed using anyconventional techniques. For example, perforations 836 can be formed aspart of the manufacturing process for the sheet or can be subsequentlyproduced by punches or other techniques. In one embodiment, one or morelasers can be used to form perforations 836. An advantage of using alaser is that perforations 836 can be formed at precise locations andwith a precise diameter so that bubbles can be formed having a precise,predefined size. Furthermore, when a laser is used to form aperforation, the material melted by the laser gathers around theperimeter edge of the perforation, thereby reinforcing the perforationand helping to prevent rupture of the sheet.

In one embodiment of the present invention, a manifold can be used forcontrolling the gas flow to one or more of spargers 804, 806, 808, 810.For example, depicted in FIG. 8 is one embodiment of a manifold 664incorporating features of the present invention. Manifold 664 comprisesa body 838 having a gas inlet port 840 and a plurality of gas outletports 842, 844, 846, 848. Gas outlet ports 842, 844, 846, 848 are inparallel communication with gas inlet port 840 by way of a forked flowpath 850. A gas source, such as a compressor or a canister of compressedgas, is fluid coupled with gas inlet port 840. The gas can be air,oxygen, or any other gas or combination of gases. Gas lines 706 extendfrom gas outlet ports 842, 844, 846, 848, respectively, to acorresponding openings 828 at first end 830 of each sparger 804, 806,808, 810, respectively. Gas lines 706 can be welded between the firstsheet 652 and the second sheet 658 at openings 828 so as to sealopenings 828 closed. Gas lines 706 can comprise flexible or rigid tubesand can be integrally formed with or separately attached to body 838.

Valves 852 are mounted on body 838 and control the flow of gas to eachgas line 706, respectively. In one embodiment, valves 852 can beelectrical valves, such as solenoid valves, that can be used to open,close, or restrict the flow of gas to spargers 804, 806, 808, 810. Inthis embodiment, electrical wiring 854 can couple to valves 852 forcontrolling their operation. In other embodiments, valves 852 cancomprise valves that are operated manually, hydraulically,pneumatically, or otherwise. By using manifold 664, different spargersor different combinations of spargers can be used at different times tooptimize performance as discussed above.

FIG. 9 illustrates a mixing system 900 according to various embodiments.The mixing system 900 may comprise a flexible compartment 902 having afirst end 904, a second end 906, and a sidewall 908, an offset driveshaft 910 being disposed within the flexible compartment 902 and havinga first bearing assembly 912 and a second bearing assembly 914 with afirst impeller 916 affixed to the first end 904 and the second end 906of the flexible compartment 902 respectively and a first impeller 916and a second impeller 918 affixed to the drive shaft 910 where theflexible compartment 902 further includes a sparger 920 having at leastone perforation 922 designed to release bubbles 924 into a fluid 926.

In various embodiments, the sparger 920 releases a gas into the fluid926 to add dissolved oxygen to the fluid 926. The location of thesparger 920 on the second end 906 of the flexible compartment 902 may beselected so that the gas bubbles 924 optimally interact with the firstimpeller 916 and the second impeller 918. For example, if a bioreactionrequires a specified amount of dissolved oxygen the mixing system 900can be optimized for residence time of the bubble 924 within the fluidby re-entrainment of the bubbles 924 by the impellers 916, 918. This canbe accomplished by adjusting the position of the sparger 920 so that allthe bubbles 924 are re-entrained, none of the bubbles 924 arere-entrained, or a specific percentage of the bubbles 924 arere-entrained. For example, the sparger 920 may be moved further awayfrom the second bearing assembly 914 which will make it less likely forthe gas bubbles 924 to be re-entrained. In various embodiments, 0%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the gas bubbles 924are re-entrained once by the first impeller 916 and/or second impeller918 or in any combination. In various embodiments, the rate of impellerrotation may be adjusted to impact the amount of re-entrainmentoccurring in the mixing system 900. The selection of bubble 924 diameterand impeller properties in combination may also impact the residencetime of the bubbles 924.

In various embodiments, the diameter and quantity of the perforations922 can be selected to optimize the operational characteristics of themixing system 900 based on the cell type being grown. For example, ifresidence time needs to be increased in a mixing system 900 to achieve adesired dissolved oxygen content in the fluid 926 the perforations 922may be smaller to create smaller gas bubbles 924. Residence time mayalso increase if the sparger 920 location and properties are selected inconjunction with the location of the impellers on the drive shaft 910for more or less re-entrainment as required by the desired bioreaction.

In various embodiments, a scalable mixing system 900 may include two ormore mixing systems 900 as shown in FIG. 9 where the systems may vary involume. The volumes may range between a test size of less than 50 litersand a commercial size of greater than 50 liters. The volumes may beselected based on scale up/down cost. For example, the optimalconditions for producing a product using a given cell type may bedetermined in the smaller test volume size so that outcomes can bepredicted in expensive commercial systems without having to expend theresources on the larger volume reaction for optimization. In someembodiments, the impeller tip speed (shear) and power input per volumemay be kept constant between the two or more mixing systems 900 whilethe quantity of impellers and their diameters may vary (See FIG. 23). Insome embodiments the sparger 920 location and number and size of theperforation 922 may vary between the test size and the commercial sizemixing systems 900.

FIG. 10 illustrates a mixing system 1000 according to variousembodiments. The mixing system 900 may comprise a flexible compartment902 having a first end 904, a second end 906, and a sidewall 908, anoffset drive shaft 910 being disposed within the flexible compartment902 and having a first bearing assembly 912 and a second bearingassembly 914 with a first impeller 916 affixed to the first end 904 andthe second end 906 of the flexible compartment 902 respectively and afirst impeller 916 and a second impeller 918 affixed to the drive shaft910 where the flexible compartment 902 further includes a sparger 920having at least one perforation 922 designed to release bubbles 924 intoa fluid 926.

In various embodiments, the drive shaft 910 may be both angled andoffset within the flexible compartment 902. Such a configuration willchange the operational parameters of the bioreaction by altering theamount of bubble 924 re-entrainment that occurs. For example, if thesparger 920 is positioned near the second bearing assembly 914 the firstimpeller 916 may re-entrain the bubbles 924 while the second impeller918 may not contact as many bubbles 924 for re-entrainment. Therefore,the dissolved oxygen content within the fluid 926 can be altereddepending on the angle of the drive shaft 910. Additionally, thebaffling effects of the rectangular shaped flexible compartment 902 canbe increased, decreased, or mixing patterns completely altered based onthe amount of offset and angled of the drive shaft 910 within theflexible compartment 902.

FIG. 11 illustrates a mixing system 1100 including systems and methodsfor gas stream mass transfer according to various embodiments. Althoughgas stream mass transfer is primarily discussed herein with regard tooxygenating a biological culture, the same methods and systems can alsobe used for oxygenating other types of liquids, such as those mentionedabove. In addition, as discussed below in greater detail, the inventivemethods and systems are not limited to oxygenating a fluid but can beused with other gases for affecting any type of mass transfer into aliquid and/or out of a liquid.

Gas stream mass transfer has a number of processing advantages when itis used for oxygenating a biological culture within a reactor container,particularly over conventional sparging techniques. Where a reactorcontainer is being designed to process a culture of cells ormicroorganisms over a relatively large change in fluid volume, thediameter of the container typically needs to be relatively large tomaintain geometry and height requirements. As the diameter of thecontainer increases with respect to volume, the depth of the culturewithin the container decreases. As a result, for very small volumes ofculture within the container, such as when the initial volume of cultureis transferred into the container, the resident time for the oxygenatingbubbles that are typically sparged into the culture from the floor ofthe container is insufficient to properly oxygenate the culture. Thatis, because the depth of culture is so shallow, the oxygenating bubblesare not within the culture for a sufficient period of time to fullyoxygenate the culture as the bubbles travel from the sparger to the topsurface of culture. Likewise, the resident time for the larger spargedbubbles used to strip out the CO₂ is also insufficient to fully removethe unwanted CO₂ from the culture. This problem is further compounded bythe fact that the CO₂ gas is heavier than air so that the CO₂ lays likea blanket over the top surface of the culture, thereby further hamperingoxygenation of the culture and removing CO₂.

In contrast to sparging which becomes more efficient as the depth of theculture increases, gas stream oxygenation or mass transfer, which isaccomplished by blowing a stream of air or other gas containing oxygenover the top surface of the culture, become more efficient as the depthof the culture or other fluid being processed decreases. Thus, gasstream oxygenation is particularly useful for shallow depth culturesdisposed within a reactor; including reactors that start with a smallvolume and increase to a large volume. In addition, sparging is known toproduce unwanted foam on the top surface of cultures, especially whenthe spargers used generate very small bubbles (sub millimeter diameter).In contrast, gas stream mass transfer produces minimal foaming and canassist in the reducing the vessel foam generation by reducing the amountof traditional sparging required. Furthermore, gas stream oxygenationprevents the formation of a CO₂ blanket on the surface of the culture.As such, the gas on the surface of the culture is both well controlledand well mixed, permitting the CO₂ to dissipate out of the culture, mixinto the head space of the reactor, and leave via the system exhaustport. The interaction of the gas stream oxygenation with the systemliquid also helps directly facilitate stripping CO₂ from the culture.Accordingly, for relatively shallow depth cultures, gas streamoxygenation can be used to both oxygenate the culture and remove CO₂from the culture, in some cases eliminating the need for traditionalsparging in certain forms of the invention.

As the depth of a culture within a reactor increases, the efficiency ofoxygenating the culture at the bottom of the reactor through gas streamoxygenation decreases. Accordingly, as the depth of the cultureincreases, dissolved O₂ sensors or other parameters or mechanisms can beused to determine when sparging or other methods of oxygenation shouldbe activated. That is, as the depth of the culture increases, spargingcan be activated such as through stepped increments or throughcontinuous gradual increase so as to ensure that the culture is alwaysproperly oxygenated. The applied gas stream oxygenation can decrease assparging increases or can remain constant. Even if the gas stream is notfully oxygenating the culture, the gas stream is still equilibrating theupper region of the culture and preventing CO₂ blanketing which in turnassists in traditional sparge operation. Thus, even for relatively deepvolumes of culture, gas stream oxygenation can continue to be used inconjunction with sparging or other methods of oxygenation. It should beappreciated that an electronic controller could be used to automaticallyactivate and/or regulate sparging and gas flow based on sensor readings.

Examples of systems will now be discussed that can be used in performinggas stream oxygenation/mass transfer. Additional examples of headspaceair flow devices and systems that can be used in the presently disclosedinvention are disclosed in U.S. Pat. No. 9,388,375 that issued on Jul.12, 2016 which is hereby incorporated by specific reference. Depicted inFIG. 11 is one embodiment of a reactor system 10 incorporating featuresof the present invention. In general, reactor system 10 comprises asupport housing 12 that bounds a chamber 14, a container assembly 16disposed within chamber 14 and a mixing system 17 coupled with containerassembly 16. Support housing 12 typically comprises a rigid tank, suchas a metal tank. The tank can be jacketed for controlling thetemperature of the culture within container assembly 16. Support housing12 can be any desired size, shape, or configuration that will properlysupport container assembly 16, as discussed below.

With continued reference to FIG. 11, container assembly 16 comprises acontainer 18 having a side 20 that extends from an upper end 22 to anopposing lower end 24. Upper end 22 terminates at an upper end wall 33while lower end 24 terminates at a lower end wall 34. Container 18 alsohas an interior surface 26 that bounds a compartment 28. Compartment 28is configured to hold a fluid. The fluid can comprise a biologicalculture which comprises cells or microorganisms, media, and othernutrients and additives. Any other type of fluid can also be used thatrequires mass transfer with a gas. For example, the fluid can be achemical, biological fluid, food product, or other fluid. For theexample herein, the fluid will be discussed as biological culture 29.Culture 29 has a top surface 31. A head space 37 is disposed withincompartment 28 and is bounded between top surface 31 of culture 29 andupper end wall 33.

In the embodiment depicted, container 18 comprises a flexible bag thatis comprised of a flexible, water impermeable material such as alow-density polyethylene or other polymeric sheets or film having athickness in a range between about 0.1 mm to about 5 mm with about 0.2mm to about 2 mm being more common. Other thicknesses can also be used.The material can be comprised of a single ply material or can comprisetwo or more layers which are either sealed together or separated to forma double wall container. Where the layers are sealed together, thematerial can comprise a laminated or extruded material. The laminatedmaterial comprises two or more separately formed layers that aresubsequently secured together by an adhesive. Examples of extrudedmaterial that can be used in the present invention include the HyQ CX3-9and HyQ CX5-14 films available from HyClone Laboratories, Inc. out ofLogan, Utah. The material can be approved for direct contact with livingcells and be capable of maintaining a solution sterile. In such anembodiment, the material can also be sterilizable such as by ionizingradiation. Prior to use, container assembly 16 is typically sealedclosed and sterilized so that compartment 28 is sterile prior to theintroduction of culture 29.

In one embodiment, container 18 can comprise a two-dimensional pillowstyle bag. In another embodiment, container 18 can be formed from acontinuous tubular extrusion of polymeric material that is cut tolength. The ends can be seamed closed or panels can be sealed over theopen ends to form a three-dimensional bag. Three-dimensional bags notonly have an annular sidewall but also a two dimensional top end walland a two dimensional bottom end wall. Three dimensional containers cancomprise a plurality of discrete panels, typically three or more, andmore commonly four or six. Each panel is substantially identical andcomprises a portion of the sidewall, top end wall, and bottom end wallof the container. Corresponding perimeter edges of each panel are seamedtogether. The seams are typically formed using methods known in the artsuch as heat energies, RF energies, sonics, or other sealing energies.

In alternative embodiments, the panels can be formed in a variety ofdifferent patterns. Further disclosure with regard to one method ofmanufacturing three-dimensional bags is disclosed in United StatesPatent Publication No. US 2002-0131654 A1, published Sep. 19, 2002,which is incorporated herein by specific reference in its entirety.

It is appreciated that container 18 can be manufactured to havevirtually any desired size, shape, and configuration. For example,container 18can be formed having a compartment sized to 10 liters, 30liters, 100 liters, 250 liters, 500 liters, 750 liters, 1,000 liters,1,500 liters, 3,000 liters, 5,000 liters, 10,000 liters or other desiredvolumes. The size of the compartment can also be in the range betweenany two of the above volumes. Although container 18 can be any shape, inone embodiment container 18 is specifically configured to be generallycomplementary to chamber 14 of support housing 12 in which container 18is received so that container 18 is properly supported within chamber14.

Although in the above discussed embodiment container 18 is depicted as aflexible bag, in alternative embodiments it is appreciated thatcontainer 18 can comprise any form of collapsible container orsemi-rigid container. In still other embodiments, container 18 can berigid and support housing 12 can be eliminated.

Continuing with FIG. 11, formed on container 18 are examples of aplurality of different ports that can be mounted thereon with each ofthe ports communicating with compartment 28. Specifically, mounted onupper end wall 33 are access ports 40 and 41 having lines 39A and Bcoupled therewith, respectively. Access ports 40 and 41 can be used fordelivering gas, media, cultures, nutrients, and/or other components intocontainer 18 and can be used for withdrawing culture 29 or gas fromwithin head space 37. For example, in some forms of the invention, port40 can be used as a gas inlet into head space 37 and port 41 can be usedas a gas outlet from head space 37. Any desired number of access portscan be formed on container 18. A sensor port 42 is formed on side 20 ofcontainer 18. A sensor 50 is disposed within sensor port 42 so as tocommunicate with compartment 28, typically at the lower end thereof. Itis appreciate that any number of sensor ports 42 can be formed oncontainer 18 each having a corresponding sensor 50 disposed therein.Examples of sensors 50 that can be used include temperatures probes, pHprobes, dissolved oxygen sensors, carbon dioxide sensors, cell masssensors, nutrient sensors, and any other sensors that allow for testingor checking the culture or production. The sensors can also be in theform of optical sensors and other types of sensors.

Mounted on lower end wall 34 are sparging ports 43 and 44. A firstsparger 52 is mounted to port 43 and is designed to deliver smallbubbles to culture 29 for oxygenating culture 29. Sparger 52 can beformed integral with or attached to port 43. A second sparger 54 ismounted to port 44 and is designed to deliver larger bubbles to culture29 for stripping CO₂ from culture 29. As such, the bubbles from firstsparger 52 are smaller than the bubbles from second sparger 54. In someforms of the invention, second sparger 54 can be an open tube or a tubewith a porous frit with relatively large pores, while first sparger 52can be a tube with a porous frit with relatively small pores. Firstsparger 52 can also comprise a perforated or porous membrane that ismounted on the end of port 43 or on the interior surface of lower endwall 34 so as to extend over port 43. It is appreciated that spargerscome in a variety of different configurations and that any type ofspargers can be used as desired or as appropriate for the expectedculture volumes, cells and conditions.

It is again noted that container 18 can be formed with any desirednumber of ports and that the ports can be formed at any desired locationon container 18. The ports can be the same configuration or differentconfigurations and can be used for a variety of different purposes suchas listed above but not limited thereto. Examples of ports and howvarious probes, sensors, and lines can be coupled thereto is disclosedin United States Patent Publication No. 2006-0270036, published Nov. 30,2006 and United States Patent Publication No. 2006-0240546, publishedOct. 26, 2006, which are incorporated herein by specific reference intheir entirety. The ports can also be used for coupling container 18 tosecondary containers, to condenser systems, and to other desiredfittings.

Also disposed alongside 20 of container 18 are a plurality of verticallyspaced apart gas ports 45-47. Each of ports 45-47 forms part of acorresponding gas delivery system, which systems are designed fordelivering gas into compartment 28 to produce gas streamoxygenation/mass transfer. Depicted in FIG. 12 is an enlarged view of agas delivery system 1200, 60A which includes gas port 45. Port 45comprises a flange 62 mounted to container 18 and a tubular stem 64outwardly projecting therefrom. Stem 64 bounds a passageway 66longitudinally extending there through so as to communicate withcompartment 28. An annular barb 68 is formed on the free end of stem 64and couples with a tube 70. In turn, tube 70 couples with an asepticconnector 72.

Aseptic connector 72 includes a first connector portion 74 thatselectively mates with and fluid couples to a second connector portion76. A tubular stem 75 projects from first connector portion 74 and fluidcouples with tube 70. Each of connector portions 74 and 76 have asealing layer 78A and B, respectively, that covers the opening toconnector portions 74 and 76. After connector portions 74 and 76 arecoupled together, sealing layers 78A and B are pulled out from betweenthe connector portions so as to form an aseptic fluid connection betweenconnector portions 74 and 76. Aseptic connectors are known in the art.One example of an aseptic connector is the KLEENPACK® connector producedby the Pall Corporation. The PALL connector is described in detail inU.S. Pat. No. 6,655,655, the content of which is incorporated herein byreference in its entirety. Other aseptic connectors can also be used.

A tube 80 fluid couples with second connector portion 76 and extends toa gas supply 82. Gas supply 82 delivers a gas which passes throughaseptic connector 72, port 45 into compartment 28. The gas can be oxygenor it can be a gas containing oxygen, such as air. Other gases can alsobe used depending on the desired application. Gas supply 82 can comprisea pressurized canister, a compressor, or other gas supply source.Disposed along tube 80 is a gas filter 84 that sterilizes the gas as itpasses there through. Also mounted along tube 80 is a valve 86. Valve 86is used to selectively stop the flow of gas through delivery system 60Aand to prevent culture 29 within container 18 from flowing out throughdelivery system 60. Valve 86 can have a variety of differentconfigurations. For example, valve 86 can comprise a ball valve, a gatevalve, a clamp that pinches tube 80 or any other type of valve thatfunctions for the intended purpose. Valve 86 can be manually controlledor can be electric, hydraulic, pneumatic or the like. It is appreciatedthat valve 86 can be positioned anywhere along delivery system 60 but istypically located close to gas port 45. In one embodiment, valve 86 canbe mounted on tube 70 adjacent to port 45 or directly on port 45.

As previously discussed, the object of gas delivery system 60 is todeliver a stream of gas over top surface 31 of culture 29 or otherapplicable fluid at a sufficient velocity and direction so that the gasstream produces a turbulence on top surface 31 that is sufficient tooxygenate the culture for growing the cells or microorganisms therein.The term “over” is broadly intended to include the gas traveling overtop surface 31 in any desired orientation such as horizontal,substantially horizontal, downwardly inclined, or upwardly inclined. Thegas stream need not flow in a linear path but can flow in a circularpath or vortex, such as about a vertical or horizontal axis, or can flowalong a random path. The gas stream can be a laminar flow or a turbulentflow and the direction, flow rate, and/or speed of the gas flow can beconstant or variable. For example, the gas stream can change from adownward vertical direction to a substantially horizontal direction. Byplacing gas port 64 on side 20 of container 18, the gas passing outthrough passageway 66 in this embodiment travels horizontally orsubstantially horizontally within compartment 28 so that it can passover and across top surface 31. In some embodiments, the gas streamoxygenation can be sufficient to independently oxygenate the culture tothe extent needed for growing the cells or microorganisms without anyother form of oxygenation, such as sparging. In other embodiments, thegas steam oxygenation can be used in conjunction with sparging or otheroxygenation processes.

In one embodiment, the gas stream oxygenation is able to achieve a masstransfer of oxygen using only air and without the aid of sparging havinga kLa factor that is greater than 3 and more commonly greater than 5 or7. The gas stream oxygenation can also maintain, without separatesparging, a stable oxygen concentration set point within the activeculture that is in a range of 30%-50% of air saturation. The abovevalues can be achieved in a stirred tank reactor with mixing by impellerand in other types of rectors. In one specific example, gas streamoxygenation, using only air, was able to oxygenate a CHO culture at atarget value of 50% of air saturation (868 mbar ambient pressure) andstrip CO₂ to a cell concentration of 3.5 E+06 cell/mL at ⅕^(th) vesselvolume. At this point the culture was then fed media to full vesselvolume. It is worth noting that the oxygenation and CO2 strippingprovided by the gas stream oxygenation was excessive at this level ofculture density and vessel fill volume; it required the addition of N₂and CO₂ mixed in with the air to hold target pH and dissolved O₂ targetvalues.

During operation, compartment 28 of container 18 is filled with culture29 so that top surface 31 is disposed close to passageway 66. In oneembodiment, the distance D₁ between passageway 66 and top surface 31 isin a range between about 0.75 cm to about 15 cm with about 1 cm to about10 cm or about 2 cm to about 5 cm being more common. Other distances canalso be used. Furthermore the distance D₁ can vary based upon factorssuch as the size of container 18, the projection angle of the gas (withflow perpendicular to the liquid surface being optimal), the flow rateof the gas, and the superficial velocity of the gas. When measuring thedistance D₁, top surface 31 can be the maximum liquid wave height underagitation of culture 29 or can be top surface 31 with no agitation. Forscalable representation, the flow rate can be measured in rate of VesselVolumes per Minute (VVM) of the maximum rated liquid working volume ofthe system. The flow rate of the gas passing out through passageway 66is typically in a range between about 0.06 VVM to about 0.2 VVM withabout 0.08 VVM to about 0.1 VVM or about 0.16 VVM to about 0.18 VVMbeing more common. Other flow rates can also be used depending on theintended application. The velocity of the gas exiting passageway 66 ortraveling across top surface 31 within compartment 28 is typically in arange between about 25 m/sec to about 275 m/sec with about 25 m/sec toabout 175 m/sec or about 30 m/sec to about 100 m/sec being more common.The velocity can be greater than 25 m/sec and more commonly greater than40 m/sec, 60 m/sec, 80 m/sec, or 100 m/sec. To achieve desired gasvelocities exiting passageway 66, passageway 66 can have a minimum exitarea of flux based on the volume of compartment 12, i.e., vessel volume(VV). This minimum exit area of flux can be in a range between about VV(liters)/80 (liters/mm²) to about VV (liters)/7.8 (liters/mm²) withabout VV (liters)/40 (liters/mm²) to about VV (liters)/30 (liters/mm²)or about VV (liters)/8.5 (liters/mm²) to about VV (liters)/6.25(liters/mm²) being more common. Other areas can also be used.

If desired, port 45 can be configured so that during operation stem 64is angled so that the gas passing out therethrough is directed slightlydown towards top surface 31. For example, stem 64 has a centrallongitudinal axis 88. Port 45 can be formed so that axis 88 of stem 64is tilted relative to horizontal during use by an angle a in a rangebetween 1° to about 10° so that the gas passing out therethrough passesslightly down against top surface 31. Other angles can also be used.

As previously discussed, gas stream oxygenation is most efficient forshallow depths of culture 29 within container 18. In one embodiment, themaximum distance D₂ (See FIG. 11) between top surface 31 and lower endwall 34 at which the gas stream oxygenation can independently oxygenateculture 29 to grow cells or microorganisms can be in a range ofdistances based on diameter of the container 18, i.e., vessel diameter(VD). For example, maximum distance D₂ can be in a range between aboutVD (cm)*0.3 to about VD (cm)*0.4. Where container 18 does not have acircular transverse cross section, VD can be based on an averagediameter. In some specific examples, D₂ can be in a range between about5 cm to 30 cm or between 10 cm and 100 cm depending on the diameter ofthe container. Other distances can also be used. At some depths, thesystem can operate without the use of sparging or other oxygenationsystems. In addition, for some depths desired oxygenation can beachieved throughout the culture without the use of a separate mixer dueto the natural circulation caused by the blowing gas. As the depthincreases, however, proper oxygenation of the culture requires both gassteam oxygenation and a separate mixing system, such as thorough animpeller or rocking, to ensure all of the culture is properlyoxygenated.

As the depth of culture 29 increases, sensors 50 may detect the need foradditional oxygenation, even when mixing is being accomplished. Anelectrical controller or manual regulator can then be used to regulatethe flow of sparged gas through spargers 52 and 54 for furthercontrolling the oxygenation and CO₂ levels within culture 29. Althoughsparging with air or oxygen may not be required at shallow depths whenusing gas steam oxygenation, sparging with nitrogen, such as throughsparger 54, may still be used at all depths to control the oxygen withinthe culture, i.e., to strip out excess oxygen produced by gas steamoxygenation. Although gas delivery system 60A is shown in FIG. 11 as theonly gas delivery system that is located at or near the elevation oncontainer 18 corresponding to the top of distance D₂, two or more gasdelivery systems 60A can be located and simultaneously operated at ornear that same elevation.

The gas delivered to container 18 through gas delivery system 60A can bedrawn out through access port 41 so that container 18 does not overinflate. Because of the rather high volume of gas passing throughcontainer 18, there can be a higher rate of evaporation of the mediarelative to conventional systems. As such, reactor system 10 can beoperated with a condenser that couples with access port 41. One exampleof a condenser that can be used with reactor system 10 is disclosed inUS Patent Publication No. 2011/0207218 A1, published Aug. 25, 2011,which is incorporated herein by specific reference in its entirety.

Culture 29 continues to grow at a level below passage 66 until a definedmass density or other desired value is determined within culture 29.Valve 86 can then be closed and media and other components added toculture 29 until the level of top surface 31 is raised to within anoperating distance from a second gas delivery system 60B shown in FIG.11. Gas delivery system 60B is then activated to again pass a gas streamover top surface 31 and thereby continue with the gas stream oxygenationof culture 29. This process can then be continued for a subsequent gasdelivery system 60C. Likewise, any number of additional gas deliverysystems can be vertically spaced apart alongside 20 of container 18 forcontinuing gas stream oxygenation at other elevations.

FIG. 13 illustrates a fluid mixing system 1300 according to variousembodiments. In various embodiments the mixing system 1300 may comprisea compartment 1302 having a drive shaft 1304 with an impeller 1306 and asparger 1308 with a gas bubble path 1310 showing the trajectory of gasbubbles rising out of the sparger 1308.

In various embodiments, the compartment 1302 includes an aspect ratio of1.5 and sparger 1308 is positioned relative to a single impeller 1306 ina position that is suboptimal for gas bubble re-entrainment. Variousapplication may require that bubbles are not re-entrained, but morefrequently re-entrainment leads to increased gas bubble residence timein the mixing system 1300 which is preferred most of the time.

FIG. 14 illustrates a fluid mixing system 1400 according to variousembodiments. In various embodiments the mixing system 1400 may comprisea compartment 1402 with a drive shaft 1404 disposed therein, affixed tothe drive shaft 1404 a second impeller 1406 and a first impeller 1408and a sparger 1410 affixed to the bottom of the compartment 1402 with agas bubble path 1412 extending therefrom.

In various embodiments, the sparger 1410 location relative to the driveshaft 1404 and first and second impeller 1406, 1408 locations issuboptimal for gas bubble re-entrainment. Such a system is sometimespreferred when additional mixing is required and there is no need toincrease bubble residence times. However, in most applications therelative locations of the sparger 1410 and impellers 1406, 1408 can beselected to alter the gas bubble path 1412 and cause longer residencetimes for the bubbles.

FIG. 15 illustrates a fluid mixing system 1400 according to variousembodiment. In various embodiments, the mixing system 1500 may comprisea compartment 1502, a drive shaft 1504 disposed within the compartment,a first impeller 1506 and a second impeller 1508 affixed to the driveshaft 1404, a sparger 1510, and a gas bubble path 1512.

In various embodiments, the relative locations of the sparger 1510 andfirst and second impellers 1506, 1508 are suboptimal for gas bubblere-entrainment. There are some applications where shorter gas bubbleresidence times are optimal, but in the majority of situations this isnot a desirable configuration.

FIG. 16 illustrates a fluid mixing system 1600 according to variousembodiments. In various embodiments, the mixing system 1600 may comprisea compartment 1602, a drive shaft 1604 disposed within the compartment1402, a first impeller 1606 and a second impeller 1608 affixed to thedrive shaft 1404, a sparger 1610, and a gas bubble path 1612 depictingthe rising flow of gas bubbles originating from the sparger 1410.

In various embodiments, the fluid mixing system 1600 of FIG. 16 isoptimized for re-entrainment. The relative positions of the sparger 1410and first and second impellers 1606, 1608 were selected to influence thegas bubble path 1612, thereby, increasing bubble residence time withinthe mixing system 1600. In the majority of applications, this is anideal arrangement.

FIG. 17 illustrates a sparger layout 1700 for experimental purposes. Thesparger layout 1700 comprises an impeller tip sweep 1702, spargerpositions 1704, and a compartment 1706.

FIG. 17 sets up an experiment where a drive shaft (not shown) is offsettoward the sidewall of a compartment 1706 and various sparger positions1704 were operated one at a time to determine optimal gas bubbledispersal patterns. In various embodiments, the corners of thecompartment 1706 act to increase gas bubble distribution by acting asbaffles.

FIGS. 18A-18F illustrate the gas bubble distribution patterns for eachof the sparger positions 1704 shown in FIG. 17. The grayed out spargerposition indicates which sparger is in operation. The other spargers areturned off. For example, sparger position 1 is in operation in FIG. 18Awhile the other sparger positions are turned off.

FIG. 19 illustrates the performance of the sparger positions 1704 shownin FIG. 17. The x-axis indicates the sparger positions 1704 in use andthe y-axis shows the time elapsed. The solid line shows the time inseconds it takes for the first released bubbles to reach the surface andthe dashed line shows the time is takes for 95% of bubbles to come tothe surface after gas flow to the sparger has ceased. Generally, havingbubbles stay in the fluid longer means that more dissolved oxygen can gointo the fluid and nurture the cells which is preferred for mostapplications. When creating a scalable system increasing or decreasingthe bubble residence time to match kLa between two or more mixingsystems will allow for better predictive models of commercial scaleproduction. As discussed previously, being able to alter the sparger andimpeller locations to change re-entrainment properties can be helpful.

FIG. 20 illustrates the performance of the sparger positions 1704 shownin FIG. 17. The x-axis indicates the sparger positions 1704 in use andthe y-axis shows the normalized kLa values obtained. Generally, a higherkLa value is preferable. In a scalable system, sparger locations can beselected to attempt to match kLa values between two or more mixingsystems having different geometries and volumes.

FIG. 21 illustrates comparison data across mixing system volumes usingthe sparging methods and systems described in this application versusprior art sparging systems. kLa is depicted on the y-axis and flowvolume per vessel volume per minute is depicted on the x-axis. It isclearly shown that prior art sparging systems and methods do not producesimilar kLa values between 50 liter to 2000 liter systems. However,spargers disclosed herein produce quite similar kLa values betweensystems having different volumes.

Using methods described above, sparger pore size and count were scaledand kLa was empirically measured comparing systems with FRIT+open pipeto systems with FRIT+DHS (sparger system and method disclosed above).

Across scales, this method improves scaled performance, reducing maximumstandard deviations of kLa across scales from 20% to 8.6% and reducingaverage standard deviation of kLa across scales employing open-pipe orDHS from 16% to 6%.

FIG. 22 illustrates mixing consistency across volumes at a given powerinput per volume. The mixing system 200 used to produce the data seen inFIG. 22 is that depicted in FIG. 2 and elsewhere in this document. Theplots shown are from data taken from a 50 liter vessel having a 2.5aspect ratio and 3 impellers, a 500 liter vessel having a 2.5 aspectratio and 3 impeller, and a 5000 liter vessel having a 2.5 aspect ratioand 3 impellers. The impeller diameters were not the same across eachscale nor the impeller diameter to vessel width ratios. As can be seen,the t95 mixing times remain constant across scales at a given power pervolume.

FIG. 23 illustrates a table showing at or near constant impeller tipspeeds and power per volume across scales 50, 100, 250, 500, 1000, and2000 liter systems. The diameter and number of impellers has changed toaccommodate different sized systems.

FIG. 24 illustrates a method of matching fluid mixing characteristicsbetween bioreactors having different volumes 2400 according to variousembodiments. Block 2402 includes the step of selecting a firstbioreactor having a set of operational parameters, the first bioreactorcomprising a first bioprocessing container having a first end, a secondend, and a sidewall and a first configurable mixing assembly suspendedbetween the first and seconds ends of the first bioprocessing container.Block 2404 includes the step of selecting a first impeller having afirst diameter. Block 2406 includes the step of attaching the firstimpeller to the configurable mixing assembly, wherein the first diameterand the attachment location are selected to conform to the operationalparameters. Block 2408 includes the step of selecting a secondbioreactor, comprising a second bioprocessing container having a firstend, a second end, and a sidewall, wherein the second bioprocessingcontainer is not the same volume as the first bioprocessing containerand a second configurable mixing assembly suspended between the firstand second ends of the second bioprocessing container. Bloc 2410includes the step of selecting a second impeller having a seconddiameter that is not the same as the first diameter. Block 2412 includesthe step of attaching the second impeller to the second configurablemixing assembly, wherein the second diameter and the second attachmentlocation are selected to match the set of operational parameters towithin five percent, wherein the set of operational parameters includepower per volume and impeller tip speed.

FIG. 25 illustrates a method of matching fluid mixing characteristicsbetween bioreactors having different volumes 2500 according to variousembodiments. Block 2502 includes the step of selecting a firstbioreactor having an operational parameters, the first bioreactorcomprising a first bioprocessing container having a first end, a secondend, and a sidewall and a first configurable mixing assembly suspendedbetween the first and seconds ends of the first bioprocessing container.Block 2504 includes the step of selecting a first sparger having a firstnumber of pores, wherein the pores have a first diameter, wherein in thefirst number and first diameter are selected to conform to theoperational parameter, wherein the first sparger is affixed to the firstend. Block 2506 includes the step of selecting a second bioreactor,comprising a second bioprocessing container having a first end, a secondend, and a sidewall, wherein the aspect ratio of the secondbioprocessing container is not the same as the aspect ratio of the firstbioprocessing container and a second configurable mixing assemblysuspended between the first and second ends of the second bioprocessingcontainer. Block 2508 includes the step of selecting a second spargerhaving a second number of pores, wherein the pores have a seconddiameter and the second sparger is affixed to the first end of thesecond bioprocessing container, wherein the second number of pores andfirst number of pores are not the same and the second diameter and firstdiameter are not the same, wherein the second number of pores and seconddiameter are selected to match the operational parameter to within fivepercent, wherein the operational parameter is kLa.

A series of predictive and actual data were collected and presented inFIGS. 26, 27, and 28. The following is a way to predict the requiredpore size to ensure scaling across bioreactors having different volumes.

1. Using empirical data and literature-based regression, predict

-   -   a. Bubble Size from pore size and flow rate (internal empirical        data regression)    -   b. Bubble terminal Velocity (Talaia, 2007, Terminal velocity of        a bubble rise in liquid column, also validated through        measurements of bubble velocities in house).    -   c. Residence time based on liquid column height divided by        bubble terminal velocity

2. kLa Score is based on:

$K_{Score} = {{\frac{{SurfaceArea}_{Single\_ bubble} \times \# {Bubbles}_{{per}\mspace{14mu} {second}} \times {ResidenceTime}}{VesselVolume}\mspace{14mu}\lbrack \text{=} \rbrack}{units}\mspace{14mu} {of}\mspace{14mu} {cm}^{2}\text{/}L}$

FIGS. 26 illustrates predictive Kscore comparison data across scales onlegacy bioreactors.

FIG. 27 illustrates actual kLa comparison data across scales on legacybioreactors.

FIG. 28 illustrates projected Kscore comparison data across scales onsome of the embodiments disclosed herein.

Using the systems and methods disclosed herein, operational parameterscan be kept constant across mixing systems accommodating differentvolumes. When moving from a test or bench scale to a commercial scalethe most import aspect is to be able to easily predict growth conditionsfor a bioreactor. The scalable system herein allows a user to createsuch a system by adjusting various aspects of the sparger design, drivesystem, and headspace airflow system where the components across systemsmay be completely different, but produce the same or similar results. Itshould be noted that all the systems interact with one another and whenselections are made the other aspects need to be considered. Forexample, if bubble residence time needs to be increased the drive systemcan altered to re-entrain bubbles. If CO2 build up needs to be reducedthen additional sparging and airflow across the headspace may beincreased. Until now a user needed to decide what metric was moreimportant (shear, power per volume, or kLa) and design scalable systemsthat were the same dimensionally (vessel aspect ratio, impeller diameterto vessel width/diameter, etc.). With the presently disclosed invention,various operational parameters can be kept constant with theunderstanding of how various systems influence one another.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Further, in describing various embodiments, the specification may havepresented a method and/or process as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process should notbe limited to the performance of their steps in the order written, andone skilled in the art will readily appreciate that the sequences may bevaried and still remain within the spirit and scope of the variousembodiments.

1-14. (canceled)
 15. A method of matching fluid mixing characteristicsbetween bioreactors having different volumes, comprising: selecting afirst bioreactor having a set of operational parameters, the firstbioreactor comprising: a first bioprocessing container having a firstend, a second end, and a sidewall; and a first configurable mixingassembly suspended between the first and seconds ends of the firstbioprocessing container; selecting a first impeller having a firstdiameter; attaching the first impeller to the configurable mixingassembly, wherein the first diameter and the attachment location areselected to conform to the operational parameters; selecting a secondbioreactor, comprising: a second bioprocessing container having a firstend, a second end, and a sidewall, wherein the second bioprocessingcontainer is not the same volume as the first bioprocessing container;and a second configurable mixing assembly suspended between the firstand second ends of the second bioprocessing container; selecting asecond impeller having a second diameter that is not the same as thefirst diameter; attaching the second impeller to the second configurablemixing assembly, wherein the second diameter and the second attachmentlocation are selected to match the set of operational parameters,wherein the set of operational parameters include power per volume andimpeller tip speed.
 16. The method of claim 15, further comprising thestep of selecting a third impeller having the third diameter that is notthe same as the first diameter and attaching the third impeller to thesecond configurable mixing assembly, wherein the third diameter and thethird attachment location are selected in combination with the seconddiameter and the second attachment location to match the set ofoperational parameters.
 17. The method of claim 16, wherein the step ofadding the third impeller reduces the required second and third impellertip speeds to maintain a power per volume and impeller tip speed in thefirst and second bioreactors.
 18. The method of claim 15, wherein theratio of the first impeller diameter to the first bioprocessingcontainer width is not the same as the ratio of the second impellerdiameter to the second bioprocessing container width.
 19. The method ofclaim 15, wherein the set of operational parameters further includesbulk fluid flow and T95 mixing times.
 20. The method of claim 19,wherein the set of operational parameters is selected based on optimalgrowth conditions for a cell.
 21. The method of claim 20, wherein thecell is eukaryotic and sensitive to a shear that increases as theimpeller tip speed increases.
 22. The method of claim 15, wherein thefirst bioprocessing container is a bench scale volume between 0.1 litersand 50 liters and the second bioprocessing container is a commercialvolume between 50 liters and 10,000 liters.
 23. The method of claim 22,wherein the first and second bioprocessing containers are rectangular inshape and the first and second configurable mixing assemblies are offsetfrom a center axis to increase bulk fluid flow.
 24. The method of claim23, wherein the aspect ratio of the first and second bioprocessingcontainers is greater than 1.5.
 25. A method of matching fluid mixingcharacteristics between bioreactors having different volumes,comprising: selecting a first bioreactor having an operationalparameter, the first bioreactor comprising: a first bioprocessingcontainer having a first end, a second end, and a sidewall; and a firstconfigurable mixing assembly suspended between the first and secondsends of the first bioprocessing container; selecting a first spargerhaving a first number of pores, wherein the pores have a first diameter,wherein in the first number and first diameter are selected to conformto the operational parameter, wherein the first sparger is affixed tothe first end; selecting a second bioreactor, comprising: a secondbioprocessing container having a first end, a second end, and asidewall, wherein the aspect ratio of the second bioprocessing containeris not the same as the aspect ratio of the first bioprocessingcontainer; and a second configurable mixing assembly suspended betweenthe first and second ends of the second bioprocessing container;selecting a second sparger having a second number of pores, wherein thepores have a second diameter and the second sparger is affixed to thefirst end of the second bioprocessing container, wherein the secondnumber of pores and first number of pores are not the same and thesecond diameter and first diameter are not the same, wherein the secondnumber of pores and second diameter are selected to match theoperational parameter to within five percent, wherein the operationalparameter is kLa.
 26. The method of claim 25, wherein the firstbioreactor includes a first headspace airflow device and the secondbioreactor includes a second headspace airflow device and each headspaceairflow device operates to provide different rates of airflow across aheadspace to match the operational parameter.
 27. The method of claim25, further comprising the step attaching a second impeller to thesecond mixing assembly, wherein the second impeller is configured tore-entrain gas bubbles rising out of the second sparger, wherein thelocation of the sparger and second impeller in combination with thesecond number and second pore size are selected to match the operationalparameter.
 28. The method of claim 25, wherein the aspect ratio of thefirst bioprocessing container is between 1.5 and 2 and the aspect ratioof the second bioprocessing container is between 1.75 and
 4. 29. Themethod of claim 25, wherein the first bioprocessing container is a benchvolume between 0.1 liters and 50 liters and the second bioprocessingcontainer is a commercial volume between 50 liters and 10,000 liters.30. The method of claim 25, wherein the first and second bioprocessingcontainers are rectangular in shape and the first and second mixingassemblies are offset from a center axis to achieve a desired kLa. 31.The method of claim 25, wherein kLa refers to O2.
 32. The method ofclaim 25, wherein kLa refers to CO2.